Method for preparing bioreactor

ABSTRACT

The present invention is a method for purifying an enzyme, particularly an acylase, comprising causing selective aggregation and precipitation of a contaminant enzyme, particularly deacetylase, by the use of a surfactant, particularly a cationic surfactant. The present invention is also a method for regenerating an immobilized enzyme carrier, particularly a synthetic adsorbent or ion exchange resin, comprising allowing a protease to act on the immobilized enzyme to remove the enzyme from the carrier, said immobilized enzyme being prepared by binding the enzyme with the carrier and optionally crosslinking the enzymes by the use of a crosslinking agent after binding. Particularly, the present invention is the above-mentioned carrier regeneration method, wherein the carrier has fine pores, and the above-mentioned carrier regeneration method, wherein the enzyme is cephalosporin C acylase.  
     According to the enzyme purification method of the present invention, undesirable contaminant enzyme for the objective enzyme, which cannot be removed by a conventional purification method, can be selectively separated and removed. Therefore, the inventive method is useful for the production of a standard enzyme product having a higher purity. In addition, the method for regenerating an immobilized enzyme carrier of the present invention based on degradation of the enzyme by a protease can remove the enzyme from the carrier, particularly a carrier having fine pores, extremely efficiently, so that repeated recycled use of the carrier is realized, which is beneficial in terms of environmental and economical aspects.

TECHNICAL FIELD

[0001] The present invention relates to a method for preparing abioreactor. More particularly, the present invention relates to a methodfor purifying an enzyme by the use of a surfactant and a method forregenerating an immobilized enzyme carrier by the use of a protease.

BACKGROUND ART

[0002] A bioreactor is a system technology to reproduce a biochemicalreaction in an artificial container, and what is meant by this term hasbeen gradually expanding. As used herein, however, a bioreactor means areactor wherein an enzyme itself is used as a catalyst. For this end,the enzyme should be immobilized in some way for an economical use of acatalyst. Thus, an immobilized enzyme plays the principal part of abioreactor. One of the important factors that determine the superiorityof an immobilized enzyme is the purity of the enzyme used.

[0003] An enzyme protein is purified from a biological sample or cellculture supernatant by appropriately combining the conventionally-knownvarious protein separation techniques according to the properties of theobjective enzyme and contaminant protein. At present, predominantseparation techniques include, for example, a method utilizingdifference in solubilities, such as salting out, solvent precipitationmethod and the like; a method utilizing difference in molecular weights,such as dialysis, ultrafiltration, gel filtration, polyacrylamideelectrophoresis and the like; a method utilizing electric charges, suchas ion exchange chromatography and the like; a method utilizing specificaffinity, such as affinity chromatography and the like; a methodutilizing difference in hydrophobicities, such as reversed phase highperformance liquid chromatography and the like; and a method utilizingdifference in isoelectric points, such as isoelectric pointelectrophoresis and the like.

[0004] In not a few cases in practice, however, contaminant proteincannot be removed completely from the objective enzyme. This isattributable to the absence of noticeable difference between theobjective enzyme and contaminant protein in various physico-chemicalproperties that the conventional protein separation methods utilize. Forexample, cephalosporin C acylase derived from Pseudomonas [enzyme thatconverts cephalosporin C and glutaryl 7-aminocephalosporanic acid(GL7-ACA) to 7-aminocephalosporanic acid (7-ACA); hereinafter to beabbreviated as CC acylase] can be purified up to an approximately 95%purity by repeatedly separating a crude cell extract solution as astarting material by steps such as dialysis, ammonium sulfatefractionation, anion exchange chromatography and the like (JapanesePatent Unexamined Publication No. 5-84078). The contaminant deacetylase,however, cannot be removed by a conventional method because it showsnearly the same behavior on anion exchange resin column as CC acylase. Adeacetylase deactylates 7-ACA to produce deacetyl 7-ACA, causing lessyield of 7-ACA. Thus, there remains a strong demand for a novel enzymepurification method capable of separating and removing such undesirableproteins.

[0005] Another factor determining the superiority of an immobilizedenzyme is the life of an immobilized enzyme carrier. In general terms,enzymes tend to be unstable to heat, strong acid, strong alkali, organicsolvent and the like, and easily lose activity even under the conditionspreferable for enzyme reactions. An immobilized enzyme shows decreasingenzyme activities with the repeated use thereof, thereby lowering theproduction efficiency of the objective substance. A degraded immobilizedenzyme is generally disposed, but when an ion exchange resin is used asa carrier, a recycled use of the carrier is desirable from environmentaland economical considerations.

[0006] Conventional methods for regenerating an immobilized enzymecarrier include use of a strong acid or strong alkali to liberate andremove the enzyme from the carrier. However, the enzyme cannot beremoved completely from the carrier by this method, which causes drasticdecrease in the activity of the immobilized enzyme upon repeatedregenerations of carrier and reimmobilizations of enzyme. Particularlywhen the carrier has fine pores, the enzyme dogs in the fine pores,resulting in appreciable degradation due to regeneration andreimmobilization.

DISCLOSURE OF THE INVENTION

[0007] It is therefore an object of the present invention to provide amethod for selectively separating and removing a contaminant enzymewhich is undesirable for the objective enzyme and which cannot beremoved by conventional separation techniques, thereby to enableproduction of a highly pure objective enzyme. Another object of thepresent invention is to provide a method for regenerating an immobilizedenzyme carrier, which is capable of efficiently removing an enzyme froma carrier.

[0008] The present inventors have conducted intensive studies in anattempt to achieve the above-mentioned objects and had a conception toutilize selective aggregation and precipitation of protein caused bysurfactants. Thus, they investigated from various aspects using thesystem of CC acylase solutions contaminated by deacetylase. The resultsrevealed that the addition of a surfactant, particularly a cationicsurfactant, leads to selective aggregation and precipitation ofdeacetylase, and using this action, the present inventors have succeededin preparing a standard CC acylase product having a high purity and ahigh production efficiency of 7-ACA.

[0009] The present inventors have also succeeded in removing CC acylaseefficiently by allowing a protease to act on an immobilized enzymeobtained by adsorbing CC acylase onto an ion exchange resin carrier andcrosslinking CC acylase with glutaraldehyde. Moreover, they have foundthat, in case of an immobilized enzyme using the above-mentioned enzymeand crosslinking agent, the use of an acidic protease is particularlyeffective for the removal of enzymes, which resulted in the completionof the present invention.

[0010] Accordingly, the present invention provides a method forpurifying an enzyme, comprising causing selective aggregation andprecipitation of contaminant enzyme by the use of a surfactant. Thepresent invention also provides the above-mentioned method for purifyingan enzyme particularly acylase, wherein the surfactant is cationic. Thepresent invention also provides a method for purifying theabove-mentioned acylase, wherein the acylase is cephalosporin C acylaseand the contaminant enzyme is deacetylase. The present invention furtherprovides a method for purifying the above-mentioned acylase, wherein thesurfactant is a methyl type or benzyl type cationic surfactant,particularly, alkyl(palm)dimethylbenzyl ammonium chloride. In addition,the present invention provides a method for purifying theabove-mentioned acylase, wherein the cationic surfactant is used in aconcentration of 0.1-0.6%.

[0011] The present invention moreover provides a method for regeneratingan immobilized enzyme carrier, comprising allowing a protease to act onan immobilized enzyme to remove the enzyme from the carrier, saidimmobilized enzyme being prepared by binding the enzyme with thecarrier, such as a synthetic adsorbent and an ion exchange resin, andoptionally crosslinking the enzymes by the use of a crosslinking agentafter binding. Particularly, the present invention provides a method forregenerating the above-mentioned carrier, wherein said carrier has finepores. Moreover, the present invention provides a method forregenerating the above-mentioned carrier, wherein the enzyme is CCacylase. Furthermore, the present invention provides a method forregenerating the above-mentioned carrier,wherein said protease is anacidic protease.

BRIEF DESCRIPTION OF THE DRAWING

[0012]FIG. 1 shows the effect of regeneration conditions on the activityof immobilized CC acylase after regeneration and reimmobilization.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The enzyme applicable to the purification method of the presentinvention is not subject to any particular limitation as long as itshows less aggregation and less precipitation than other contaminantenzymes with respect to at least one surfactant Preferably, not lessthan 70% of the enzyme remains in a solution under the conditionswherein not less than 95% of the contaminant enzyme aggregates andprecipitates. Examples of preferable enzyme include acylase,particularly CC acylase.

[0014] The contaminant enzyme to be removed by the purification methodof the present invention should have property to allow easieraggregation and precipitation than the objective enzyme with respect toat least one surfactant. For example, when the objective enzyme isacylase, the contaminant enzyme is exemplified by deacetylase and thelike. The contaminant enzyme may be contained in plurality. When eachcontaminant enzyme is aggregated and precipitated by different kinds ofsurfactants, one surfactant is added first to cause aggregation andprecipitation of a first contaminant enzyme, followed by removal of theprecipitates by centrifugation or filtration, and then, a differentsurfactant is added to cause aggregation and precipitation of anothercontaminant enzyme.

[0015] The surfactant to be used in the present invention is notparticularly limited and can be selected from among cationic, anionicand nonionic surfactants, in accordance with the property of theobjective enzyme and contaminant enzyme. When deacetylase, whichcontaminates an acylase solution, is to be removed, for example,preferably a cationic surfactant, more preferably a methyl type orbenzyl type cationic surfactant, particularly methyl and benzyl typecationic surfactants [e.g., alkyl(palm)dimethylbenzyl ammonium chlorideand the like], are used.

[0016] The surfactant can be used in a concentration that variesdepending on the kind of surfactant and the combination of the objectiveenzyme and contaminant enzyme. The desirable concentration is such that95% or more of the contaminant enzyme aggregates and precipitates, and70% or more of the objective enzyme remains in the solution. When theobjective enzyme is acylase and the contaminant enzyme is deacetylase,for example, the surfactant is added to the concentration of 0.1-0.6%.

[0017] The carrier that can be applied to the inventive method forcarrier regeneration is subject to no particular limitation as long asit can be used for immobilization of enzyme by typical carrier bindingmethods. Examples thereof include natural adsorbents such as activecharcoal, acidic clay, kaolinite, celite, alumina, bentonite, silicagel, titanium oxide, chitin, tannin and the like; synthetic adsorbentssuch as polystyrene resin having a hydrophobic residue (e.g., propyl,butyl, hexyl, phenyl and the like) introduced thereinto, polyacrylamidegel and the like; polysaccharide gel having an ion exchange group; ionexchange resin; and the like. Preferred are synthetic adsorbents and ionexchange resin. The kind of carrier can be appropriately changedaccording to the property of the enzyme to be immobilized and the like.

[0018] The inventive carrier regeneration method is particularlyeffective when the carrier has fine pores (the fine pores in the presentinvention mean those having a diameter of 100 nm or less). When anenzyme has been immobilized on a carrier by a carrier binding method anda crosslinking method in combination, for example, a conventional methodusing a strong acid or a strong alkali fails to dissolve the enzymeinsolubilized by forming crosslinks. The enzyme then cloggs in the finepores to cause striking degradation of the carrier. In contrast, theinventive regeneration method using a protease provides efficientremoval of the insolubilized enzyme from the carrier, since theinsolubilized enzyme is degraded by the action of the protease,resulting in redissolution of the enzyme.

[0019] The enzyme to be immobilized on a carrier is subject to noparticular limitation as long as it can be bound with the carrierwithout losing its activity by physical adsorption, ionic bonding andthe like, and various enzymes derived from microorganisms, plants andanimals can be used. Industrialized enzymes include acylase, aminoacylase, invertase, nitrile hydratase, sugar isomerase and the like.

[0020] An enzyme can be immobilized onto a carrier by a conventionalcarrier binding method (an immobilizing method wherein an enzyme isbound with a water insoluble carrier, including a binding method byphysical adsorption in the present invention). Particularly, an enzymeis desirably bound with a carrier by ionic bonding, hydrophobic bondingor physical adsorption. When an ion exchange resin is used as a carrier,for example, an equilibrated resin is packed in a column and a suitablebuffer containing an enzyme is passed through the column (400-1000units/mL column), thereby allowing the enzyme to bind with the resin.

[0021] When the carrier binding alone is not sufficient to preventenzyme leakage, enzymes after binding with the carrier are crosslinkedwith a crosslinking agent and immobilized. Examples of the crosslinkingagent include glutaraldehyde, hexamethylene diisocyanate and the like.The crosslinking reaction is performed by washing the immobilized enzymeobtained by the above-mentioned method and circulating a crosslinkingagent dissolved in a suitable buffer through a column for approximately0.5-2 hours. The unreacted functional group in the crosslinking agentneeds to be inactivated with an inactivating agent, such as glycine,which is circulated through the column for approximately 0.5-1 hour.

[0022] An immobilized enzyme carrier can be regenerated as in thefollowing. An immobilized enzyme prepared by the above-mentionedimmobilization method is repeatedly subjected to known enzyme reactionuntil it loses half the enzyme activity, and a solution (pH 3-5 or pH9-11) containing 3-50 units/mL of an acidic protease (or alkalineprotease) is passed through the column containing said immobilizedenzyme, in an about 5-15-fold volume of the column and at a flow rate of5-20 column volume/hour (hereinafter to be indicated as SV 5-20) forabout 1 to 5 hours. Regeneration reaction temperature is 20-60° C.,preferably 30-50° C.

[0023] The enzyme can be reimmobilized onto a regenerated immobilizedcarrier in the same manner as in the first immobilization.

EXAMPLES

[0024] The present invention is explained in more detail in thefollowing byway of Examples that are for exemplification only, and donot limit the present invention in any way.

Example 1 Separation and Removal of Deacetylase from CC Acylase Solutionby Various Surfactants

[0025] (1) Culture of CC Acylase Producing Bacteria and Purification ofCrude Extract Solution

[0026] Mutant CC acylase N-176 producing E. coli JM109 (PCCN 176-2) FERMBP-3047 was cultured according to the method disclosed in Example 3 ofJapanese Patent Unexamined Publication No. 5-84078 and the cells wereharvested by centrifugation. The obtained cell pellets were disruptedwith a homogenizer and soluble protein component was extracted withwater, which was followed by centrifugation to remove debris, whereby acrude extract solution was obtained.

[0027] (2) Removal of Contaminant Deacetylase by Surfactant

[0028] Polyethyleneimine was added to the crude extract solutionobtained in the above-mentioned (1) to allow precipitation of thenucleic acid, which was followed by centrifugation to recoversupernatant. This supernatant was applied to DEAE column equilibratedwith phosphate buffer (pH 7.0), washed with water and eluted with0.2M-NaCl. (By this step, β-lactamase, which is the other contaminantenzyme, is selectively recovered and removed in nonadsorbed Traction.)To the obtained eluate was added a cationic surfactant:alkyl(palm)dimethylbenzyl ammonium chloride [trademark : Cation F2-50;manufactured by NOF Corporation (hereinafter the same)],alkyl(palm)trimethyl ammonium chloride (trademark: Cation FB) ordodecyltrimethyl ammonium chloride [trademark: Cation BB], an anionicsurfactant: alkyl glycine [trademark: Nissan Anon LG] or a nonionicsurfactant: polyoxyethylene octylphenyl ether (trademark : NonionHS-210) or polyoxyethylenesorbitan monooleate (trademark: Nonion OT-221)to the final concentrations of 0.1, 0.3 and 0.5%, and the mixtures werestood still at room temperature for 16 hours. The activities of CCacylase and deacetylase in each sample before and after the addition ofthe surfactant were measured and the changes thereof were examined. Theenzyme activity was calculated by quantitatively determining 7-ACA anddeacetyl 7-ACA produced from GL7-ACA as a substrate. The results areshown in Table 1. Cation F2-50 showed 90% or more of CC acylase residualactivity at the concentration of 0.1-0.3% and reduced the activity ofcontaminant deacetylase to not more than 5%. At the concentration of0.5%, deacetylase was removed nearly completely. The other two cationicsurfactants also showed selective removal of deacetylase at theconcentration of 0.5%. When an anionic or nonionic surfactant was used,deacetylase could not be removed completely in the concentration rangeexamined, but the deacetylase activity decreased to nearly half or lessalmost without decrease of the CC acylase activity. TABLE 1 Effect onselective removal of contaminant deacetylase by the addition of varioussurfactants residual activity¹⁾ (CC acylase/deacetylase) SurfactantAddition concentration (%) (trademark) 0 0.1 0.3 0.5 Cation F2-50100/100 95/5  92/1  70/0  Cation FB 100/100 100/55  100/40  80/1  CationBB 100/100 100/55  100/45  85/5  Nissan Anon LG 100/100 100/60  100/50 95/30 Nonion HS-210 100/100 100/50  100/50  100/45  Nonion OT-221100/100 100/50  100/50  100/45 

Example 2 Dissolution of Crosslinked CC Acylase Precipitate by Protease

[0029] To 10 mM Tris HCl buffer (pH 8.0, 5 mL) containing 67 units/mL ofCC acylase N176 (how to obtain this enzyme is disclosed in JapanesePatent Unexamined Publication No.5-84078) was added 150 mM Tris HClbuffer (pH 8.7, 5 mL) containing 20% glutaraldehyde and the mixture wasstirred for 1 hour to allow reaction, whereby a crosslinked product wasprecipitated. The supernatant was removed by centrifugation and theprecipitate was recovered. To the precipitate was added 2N—NaOH,0.1N—NaOH, 0.1N—NaOH+alkaline protease [Bioprase AL-15 (30 units);manufactured by Nagase Biochemicals Ltd.], 2N—HCl, 0.1N—HCl or 0.1N—HCl+acidic protease [Denapsin (30 units); manufactured by NagaseBiochemicals Ltd.] by 3 mL and the precipitate was immersed therein for2 hours at 40° C. As a result, addition of NaOH or HCl alone did notcause change in the precipitate, but addition of HCl+acidic proteaseresulted in the complete dissolution of the precipitate and a clearsolution was obtained. While the addition of NaOH+alkaline protease alsoresulted in nearly complete dissolution of the precipitate, the solutionwas cloudy and complete dissolution was not achieved. From the aboveresults it was shown that CC acylase insolubilized by crosslinking gtreatment is effectively redissolved by the use of protease,particularly an acidic protease.

Example 3 Regeneration of Immobilized CC Acylase Carrier by Protease

[0030] (1) Preparation of Immobilization Carrier

[0031] A brand new styrene-divinyl benzene highly porous strong basicion exchange resin [80 mL, HPA 25 (having fine pores of diameter 10-100nm); manufactured by Mitsubishi Chemical Corporation] was immersed in1N—NaOH:methanol=1:1 for one day and packed in a column (350 mm×15 cm).Then, 3-fold volume of 2N—HCl was passed through the column at SV3 andthe column was washed with 5-fold volume of water (SV3), whereafter5-fold volume of 0.1N—HCl was passed through the column at SV3 and thecolumn was washed with water at SV3 until the pH at the outlet of thecolumn became 4 or above.

[0032] (2) Immobilization of CC Acylase

[0033] HPA 25 (80 mL) prepared in the above-mentioned (1) was packed ina column and 10 mM Tris HCl buffer (pH 8.0, 240 mL) containing CCacylase N176 (67 units/mL was passed through the column at SV3. Afterwashing the column with 240 mL of water (SV3), 60 mM phosphate buffer(pH 8.0, 300 mL) containing 2% glutaraldehyde was circulated at SV20 forone hour while adjusting the pH to 7.5. After crosslinking, the columnwas washed with 400 mL of water (SV3). Then, 60 mM phosphate buffer (pH8.0, 300 mL) containing 0.2M glycine was circulated at SV10 for one hourto inactivate unreacted aldehyde groups. Using water (400 mL or more),the column was washed at SV5 to give 82 mL of acylase immobilized on HPA25.

[0034] (3) Production of 7-ACA by Immobilized CC Acylase

[0035] CC Acylase immobilized on HPA 25 (20 mL) and prepared in theabove-mentioned (2) was packed in a column with a jacket, and 6.0 mg/mLglutaryl 7-aminocephalosporanic acid (GL7-ACA) solution (100 mL) wascirculated for one hour at 20° C. at circulation flow rate of about 70mL/min while adjusting the pH to 7.75 with NaOH, whereby 7-ACA wasobtained. After the reaction, the reaction mixture in the column waspushed out with water and 7-ACA was recovered. The above-mentionedreaction was repeated 100 times (continuous batch reaction), which wastaken as one cycle.

[0036] (4) Regeneration of Immobilization Carrier

[0037] Using 2N—NaOH, 0.1N—NaOH+Bioprase (3000 ppm, 10 units/mL) or0.1N—HCl+Denapsin (3000 ppm, 10 units/mL) as a regeneration agent, HPA25was regenerated every one cycle. The regeneration agent (10-fold volumeof carrier) was circulated at 40° C., SV about 20 for 5 hours.

[0038] After the regeneration reaction, the above-mentioned steps(1)-(4) were repeated 2 to 4 times, and the CC acylase activity afterreimmobilization (2) was determined. The changes of the activities werecompared.

[0039] The results are shown in FIG. 1. When 2N—NaOH was used as aregeneration agent, the activity of the immobilized CC acylase decreasedin nearly half at every cycle, whereas the use of 0.1N—HCl and Denapsin(acidic protease) did not result in decrease of the enzyme activity evenafter.3 cycles. When 0.1N—NaOH and Bioprase (alkaline protease) wereused, the activity of CC acylase decreased at every cycle but in asmaller magnitude as compared to the single use of NaOH, and not lessthan 50% of the original activity was expressed even after 4 cycles.

[0040] The enzyme purification method of the present invention ischaracterized in that undesirable contaminant enzyme for the objectiveenzyme, which cannot be removed by a conventional purification method,is separated and removed by utilizing the selective aggregation andprecipitation of protein by a surfactant. According to the presentinvention, therefore, a standard enzyme product having a higher puritythan the conventional products can be produced. In addition, the methodfor regenerating an immobilized enzyme carrier of the present inventionbased on the action of protease on enzyme can remove enzyme from acarrier extremely efficiently, so that a long term continuous recycleduse of the carrier is realized. Consequently, the present inventiongreatly contributes to the reduction of the production cost of abioreactor and prevention of environmental destruction associated withthe waste disposal of carrier.

[0041] This application is based on application Nos. 212383/1996 and212387/1996 filed in Japan, the contents of which are incorporatedhereinto by reference. All publications are herein incorporated byreference to the same extent as if each individual publication wasspecifically described herein.

What is claimed is:
 1. A method for purifying an enzyme, comprising causing selective aggregation and precipitation of a contaminant enzyme by the use of a surfactant.
 2. The method of claim 1, wherein the surfactant is cationic.
 3. A method for purifying acylase, comprising causing selective aggregation and precipitation of contaminant enzyme by the use of a cationic surfactant.
 4. The method of claim 3, wherein the acylase is cephalosporin C acylase, and the contaminant enzyme is deacetylase.
 5. The method of claim 3 or claim 4, wherein the cationic surfactant is of a methyl type or a benzyl type.
 6. The method of claim 5, wherein the cationic surfactant is alkyl(palm)dimethylbenzyl ammonium chloride.
 7. The method of any of claims 3 to 6, wherein the cationic surfactant is used in a concentration of 0.1-0.6%.
 8. A method for regenerating an immobilized enzyme carrier, comprising allowing a protease to act on an immobilized enzyme to remove the enzyme from the carrier, said immobilized enzyme being prepared by binding the enzyme with the carrier and optionally crosslinking the enzymes by the use of a crosslinking agent after binding.
 9. The method of claim 8, wherein the carrier is a synthetic adsorbent or an ion exchange resin.
 10. The method of claim 8 or claim 9, wherein the carrier has fine pores.
 11. The method of any of claims 8 to 10, wherein the enzyme is cephalosporin C acylase.
 12. The method of any of claims 8 to 11, wherein the protease is an acidic protease. 